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Chapter 8

Mycotoxins in Cereal andSoybean-Based Food and Feed

Małgorzata Piotrowska, Katarzyna Śliżewska andJoanna Biernasiak

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54470

1. Introduction

1.1. Toxigenic fungi and mycotoxins in cereal and soybean products

Cereals and soybean are plants used extensively in food and feed manufacturing as a sourceof proteins, carbohydrates and oils. These materials, due to their chemical composition, areparticularly susceptible to microbial contamination, especially by filamentous fungi. Cereals,soybean, and other raw materials can be contaminated with fungi, either during vegetationin the field or during storage, as well as during the processing.

Fungi contaminating grains have been conventionally divided into two groups – field fungiand storage fungi. Field fungi are those that infect the crops throughout the vegetationphase of plants and they include plant pathogens such as Alternaria, Fusarium, Cladosporium,and Botrytis species. Their numbers gradually decrease during storage. They are replaced bystorage fungi of Aspergillus, Penicillium, Rhizopus and Mucor genera that infect grains afterharvesting, during storage [1]. Both groups of fungi include toxigenic species. Currently,this division is not so strict.

Therefore, according to [2], four types of toxigenic fungi can be distinguished:

• Plant pathogens as Fusarium graminearum and Alternaria alternata;

• Fungi that grow and produce mycotoxins on senescent or stressed plants, e.g. F. monili‐forme and Aspergillus flavus;

• Fungi that initially colonize the plant and increase the feedstock’s susceptibility to con‐tamination after harvesting, e.g. Aspegillus flavus.

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• Fungi that are found on the soil or decaying plant material that occur on the developingkernels in the field and later proliferate in storage if conditions permit, e.g. Penicillium ver‐rucosum and Aspergillus ochraceus.

Fungal growth is influenced by complex interaction of different environmental factors suchas temperature, pH, humidity, water activity, aeration, availability of nutrients, mechanicaldamage, microbial interaction or the presence of antimicrobial compounds. Poor hygiene,inappropriate temperature and moisture during harvesting, storage, processing and han‐dling may contribute to increased contamination extent.

Fungal contamination can cause damage in cereal grains and oilseeds, including low germi‐nation, low baking quality, discoloration, off-flavours, softening and rotting, and formationof pathogenic or allergenic propagules.

It may also decrease the kernel size and thus affect the flour yield. Moulds growing on stor‐ed cereals produce a range of volatile odour compounds, including 3-octanone, 1-octen-3-ol,geosmin, 2-methoxy-3-isopropylpyrazine, and 2-methyl-1-propanol which are responsiblefor an earthy-musty off-odour and affect the quality of raw materials even when present invery small amounts [3]. Moulds produce a vast number of enzymes: lipases, proteases, amy‐lases, which are able to break down food into components leading to its spoilage. Fungigrowing on stored grains can reduce the germination rate and decrease the content of carbo‐hydrate, protein and oils. During storage of soybean seed lasting 12 months, the moisturecontent was at the level of 10-11%. It was observed that the germination rate decreased frominitial 75% to 4% prior to the lapse of a 9-month period. In prolonged storage under naturalconditions, the total carbohydrate content decreased from 21% to 16.8%, and protein and thetotal oil contents became slightly reduced [4]. Moulds as food and feed spoilage microorgan‐isms have been characterized in several review articles [2, 5].

The largest producers of soybean in the World are the United States of America, Brazil, Ar‐gentina, China, and India. The climatic conditions in soybean-growing regions (moderatemean temperature and relative humidity between 50 and 80%) provide optimal conditionsfor fungal growth. Soybean (Glyccine max L.Merr.) is often attacked by fungi during cultiva‐tion, which significantly decreases its productivity and quality in most production areas.Fungi associated with cereal grains and oilseeds are important in assessing the potential riskof mycotoxin contamination. Mycotoxins are fungal secondary metabolites which are toxicto vertebrate animals even in small amounts when introduced orally or by inhalation.

Table 1 summarises the occurrence of contamination of different raw materials in variouscountries. Some of them are of mycotoxicological interest.

Soybean matrix has been rarely studied compared to cereals in relation to fungal and myco‐toxin contamination. The fungi associated with soybean seeds, pods and flowers in NorthAmerica were reviewed by [20]. The most common species belong to Aspergillus, Fusarium,Chaetomium, Penicillium, Alternaria and Colletotrichum genera. Most of these fungi were re‐corded in mature seeds prior to storage. About 10% of them are commonly referred to asstorage moulds. Most of the isolated fungi are facultative parasites or saprophytes.

Soybean - Pest Resistance186

Commodities Country Fungal species Ref

Soybean Ecuador Aspergillus flavus, A.niger, A.ochraceus, A.parasiticus, Fusarium

verticillioides, F.semitectum, Penicillium janthinellum, P.simplicissimum,

Nigrospora oryzae, Cladosporium cladosporioides, Arthrinium

phaeospermum

[6]

Romania Aspergillus flavus, A.parasiticus, A.candidus, A.niger, Penicillium

griseofulvum, P.variabile, Fusarium culmorum, F.graminearum,

F.oxysporum

[7]

India Aspergillus flavus, A.candidus, A.versicolor, Eurotium repens,

A.sulphureus, Fusarium sp., Alternaria sp., Curvularia sp.

[4]

USA Diaporthe phaseolorum var. sojae, Fusarium sp., Alternaria alternata,

Alternaria sp., Fusarium sp., Curvularia sp., Cladosporium sp., Fusarium

equiseti, F.oxysporum, F.solani

[8-10]

Croatia Fusarium sporotrichides, F.verticillioides, F.equiseti, F.semitecium,

F.pseudograminearum, F.chlamydosporum, F.sambucinum

[11]

Argentina Aspergillus flavus, A.niger, A.candidus, A.fumigatus, Fusarium

verticillioides, F.equiseti, F.semitecium, F.graminearum, Penicillium

funiculosum, P.griseofulvum, P.canenscens, Erotium sp. Cladosporium

sp., Alternaria alternata, A.infectoria, A.oregonensis

[12, 13]

Rice Ecuador Aspergillus flavus, A.ochraceus, Fusarium verticillioides, F.oxysporum,

F.proliferatum, F.semitectum, F.solani, Penicillium janthinellum,

Epicoccum nigrum, Curvularia lunata, Nigrospora oryzae, Rhizopus

stolonifer, Bipolaris oryzae

[6]

Wheat Argentina Aspergillus flavus, A.niger, A.oryzae, Fusarium verticillioides, Penicillium

funiculosum, P.oxalicum

[12]

Germany Aspergillus candidus, A.flavus, A.versicolor, Eurotium sp., Penicillium

auriantogriseum, P.verrucosum, P.viridicatum, Alternaria sp.

[14]

Poland Alternaria tenuis, Aspergillus aculeatus, A.parasiticus, Fusarium

moniliforme, F.verticillioides, Penicillium verrucosum, P.viridicatum

P.crustosum

[15]

Croatia Fusarium graminearum, F.poae, F.avenaceum, F.verticillioides [11]

Maize Ecuador Aspergillus flavus, A.parasiticus, Fusarium graminearum,

F.verticillioides, Mucor racemosus Rhizopus stolonifer, Acremonium

strictum, Alternaria alternata, Cladosporium sp.

[6]

Poland Aspergillus aculeatus, Aspergillus parasiticus, Fusarium moniliforme,

F.verticillioides

[15]

Argentina Fusarium verticillioides, F.proliferatum, F.subglutinans, F.dlamini,

F.nygamai, Alternaria alternata, Penicillium funiculosum, P.citrinum,

Aspergillus flavus

[16, 17]

Mycotoxins in Cereal and Soybean-Based Food and Feedhttp://dx.doi.org/10.5772/54470

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Commodities Country Fungal species Ref

Croatia Fusarium verticillioides, F.graminearum [11]

Oats Poland Cladosporium sp., Aspergillus sp., Penicillium sp. [18]

Breakfast cereals Poland Aspergillus versicolor, A.flavus, A.sydowi, A.niger, A.ochraceus,

Fusarium graminearum, Penicillium chrysogenum, Eurotium repens

[19]

Wheat flour Germany Aspergillus candidus, A.flavus, A.niger, Eurotium sp. Penicillium

auriantogriseum, P.brevicompactum, P.citrinum, P.griseofulvum,

P.verrucosum, Cladosporium cladosporioides

[14]

Table 1. Fungal species dominated in cereals and cereal products

Fusarium graminearum is associated with cereals and soybean growing in warmer areas suchas South and North America or China, and F.culmorum in cooler areas such as Finland,France, Poland or Germany. Mechanical damage of kernels by birds or insects, e.g. Europe‐an corn borer and sap beetles, predisposes corn to infections caused by Fusarium and other“field fungi”. Fusarium moniliforme and F.proliferatum are the most common fungi associatedwith maize. It was found that the levels of contamination with Fusarium sp. were significant‐ly greater on the conventional than the transgenic cultivars in 2000, but in 1999 the differ‐ence between the cultivars was not statistically significant. In case of Alternaria, a greaterfrequency of contamination in transgenic varieties was observed. The authors concludedthat the isolation frequency can vary by years and is more dependent on the environmentaland cultural practices than on varieties [9]. The isolation frequencies of fungi from seeds andpods of soybean cultivars varied annually, in part due to some differences in environmentalconditions (rainfall) [8].

Fusarium species occur worldwide in a variety of climates and on many plant species as epi‐phytes, parasites, or pathogens. Fusarium-induced diseases of soybean have been attributedto different species: Fusarium oxysporum (fusarium blight, wilt and root rot), Fusarium semite‐ctum (pod and collar rot), F.solani (sudden death syndrome) [21, 22]. Fusarium infections arespread by air-borne conidia on the heads or by a systemic infection. The species belonging toFusarium genera are of particular interest due to the formation of a wide range of secondarymetabolites, many of which are toxic to humans or animals. Infections by Fusarium spp.were determined by [11] in different crops. The contamination expressed as the percentageof seeds with Fusarium colonies ranged from 5% to 69% for wheat, from 25% to 100% formaize, from 4% to 17% for soybean. The dominant species were F.graminearum on wheat(27% of isolates), F.verticillioides on maize (83 % of isolates), and F.sporotrichioides on soybean(34 % of isolates) [11]. This study suggested that the risk of contamination with Fusariumtoxins is higher for maize and wheat than for soybean.

The mycological state of grain can be considered as good when the number of CFU is with‐in the range 103-105 per gram [23]. In our research, the contamination of feed componentssuch as barley, maize and wheat was in the range from 102 to 104 CFU/g, depending on thecrop, region and mills [15]. It was found that wheat from organic farms was contaminated


with fungi by 70.5% more and barley by 24.8% less as compared to the crops from conven‐tional farms [24]. Similarly, the total number of fungi in Polish ecological oat products wasabout a hundred times higher than in conventional ones. In samples of ecological origin,the mean value of fungi was 1.1×104 CFU/g, whereas for conventional grains it was 5.0×102

CFU/g [18].

The results obtained by [14] showed that the most common moulds isolated from wholewheat and wheat flour belong to the Aspergillus and Penicillium genera. From the wholewheat flour, 83.7% of Aspergillus followed by Penicillium (7.6%), Eurotium (2.9%) and Al‐ternaria (2.5%) species were isolated. The white flour contained 77.3% of Aspergillus, 15%of Penicillium and 4.1% of Cladosporium genera. Aspergillus candidus was the dominantspecies. Among all the isolated fungal species, 93.2% belonged to the group of toxigenicfungi. Several toxin-producing Aspergillus species were reported to dominate on cereals,especially A.flavus, A.candidus, A.niger, A.versicolor, A.penicillioides, and Eurotium sp. atlower water activity [25]. Among Aspergillus species isolated from Ecuadorian soybeanseeds, Aspergillus flavus and A.ochraceus were the most prevalent ones. The most frequentFusarium species were F.verticillioides and F.semitecium. All the examined samples werecontaminated with these species [6]. The presence of mycobiota in raw materials and fin‐ished fattening pig feed was determined in eastern Argentina. All samples of soybeanseeds were contaminated with fungi in the range from 10 to 9.0×102 CFU/g, dependingon the sampling period. The most prevalent species in soybean and wheat bran were As‐pergillus flavus and Fusarium verticillioides [12].

The fungal microflora changes during post-harvest drying and storage. The field fungi areadapted to growth at high water activity and they die during drying and storage, to be re‐placed by storage fungi that are capable of growing at lower aw. For most grains, moisturecontent in the range from 10% to 14% is recommended, depending on the grain type anddesired storage life [1].

A wide range of microorganisms have been isolated from storage grains, including psychro‐tolerant, mesophilic, thermophilic, xerophilic and hydrophilic species. The extremely xero‐philic species are Eurotium spp. and Aspergillus restrictus, the moderate xerophilic onesinclude A.candidus and A.flavus, and the slighty xerophilic one is A.fumigatus. An example ofpsychrotolerant species belonging to Penicillium genera is P.aurantiogriseum and P.verruco‐sum, mesophilic species can be represented by P.corylophilum, and thermophilic species byTalaromyces thermophilus. Among the hydrophiles, the most common are Fusarium and Acre‐monium species [25]. The minimum aw for conidial formation is influenced by temperature,for instance, P.aurianogriseum produces conidia to a minimum of 0.86 aw at 30oC, but to 0.83aw at 23oC. Many species belonging to Aspergillus and Penicillium genera are highly adaptedto the rapid colonisation of substrates of reduced water activity. Modifying several factors ingrain storage may facilitate safe storage. Stores should be monitored for relative humidity,temperature and airflow efficiency. Moisture migration may occur during storage and createdamp pockets. In addition to this, insect infestations may cause heating and the generationof moisture. Aeration with cool air may help to protect the stored commodities against fun‐gal development.


189

2. Conditions affecting mycotoxin production

Cereals in the field are exposed to fungi from the soil, birds, animals, insects, organic fertil‐izers, and from other plants in the field. Mechanical damage of raw material or food due toinsects and pests is a disturbing problem mainly in tropical regions, particularly as foodcontaminants are present in the field more abundantly than in the storage. Many differentinsects, e.g. European corn borer and sap beetles have the capability of promoting infectionsof various crops with mycotoxigenic fungi [25].

Mycotoxin production is determined by genetic capability related to strain and environmen‐tal factors including the substrate and its nutritious content. Toxin production is dependenton physical (temperature, moisture, light), chemical (pH value, nutrients, oxygen content,preservatives), and biological factors (competitive microbiota). Each fungus requires specialconditions for its growth and other conditions for its toxin production.

2.1. Physical factors

The most important factor governing colonisation of grains and mycotoxin production is theavailability of water which on the field comes mainly with rainfall. The second important fac‐tor is temperature. The moisture and temperature effects on mycotoxin production often dif‐fer from those on germination and growth. Table 2 presents the moisture and temperaturerequirements of most common toxigenic fungi for their growth and mycotoxin production.

It was found that optimal temperature for F.graminearum growth on soybean contained inthe range 15-20oC (in isothermal temperature) and 15/25oC (in cycling temperature). The op‐timal temperature for mycotoxin production on soybean was 20oC for deoxynivalenol(DON) and 15oC for zearalenone (ZEA). After 15 days of incubation, the maximum levels 39ppm and 1040 ppm for ZEA and DON, respectively, were detected. Fumonisins were pro‐duced by Fusarium graminearum only the on culture medium at 30oC; on soybean no fumoni‐sins were detected [31].

Most fungi need at least 1-2% of O2 for their growth. The influence of high carbon dioxideand low oxygen concentrations on the growth and mycotoxin production by the foodbornefungal species was investigated by [32]. Three groups of species were distinguished: first,which did not grow in 20% CO2 <0.5% O2 (Penicillium commune, Eurotium chevalieri and Xero‐myces bisporus); second, which grew in 20% CO2 <0.5% O2, but not 40% CO2 <0.5% O2 (Peni‐cillium roqueforti and Aspergillus flavus); and third, which grew in 20%, 40% and 60% CO2

<0.5% O2 (Mucor plumbeus, Fusarium oxysporum, F.moniliforme, Byssochlamys fulva and B.ni‐vea). The production of aflatoxin, patulin, and roquefortine C was greatly reduced under allof the atmospheres tested. For example, aflatoxin was not produced by A. flavus duringgrowth under 20% CO2 for 30 days. Patulin was produced by B.nivea in the atmospheres of20% and 40% CO2, but only at low levels [32].

2.2. Chemical factors

Nutritional factors such as carbonohydrate and nitrogen sources and microelements (cop‐per, zinc, cobalt) affect mycotoxin production, but the mechanisms of this impact are still


unclear. A relationship between mycotoxin production and sporulation has been document‐ed in several toxigenic fungi. For example, chemical substances that inhibit sporulation ofAspergillus parasiticus have also been shown to inhibit the production of aflatoxin [33].Chemical preservatives such as organic acids (sorbic, propionic, acetic, benzoic) or fungi‐cides have been used to restrict the growth of mycotoxigenic fungi. It was found that pro‐pionic acid at the concentration of up to 0.05% inhibited the growth and ochratoxinproduction by Penicillium auriantogriseum. A more effective result in higher temperature wasobserved [34]. Inhibiting fungal growth and toxigenic properties by organic acids is connect‐ed with lowering the pH value. It was found that ammonium and sodium bicarbonate at theconcentration of 2% fully inhibited the development of the cultures of Aspergillus ochraceus,Fusarium graminearum and Penicillium griseofulvum inoculated into corn. The production ofochratoxin A by Aspergillus ochraceus was reduced from 26 ppm in untreated corn to 0.26ppm in bicarbonate-treated corn samples [35].

2.3. Biological factors

The simultaneous presence of different microorganisms, such as bacteria or other fungi,could disturb fungal growth and the production of mycotoxins. For instance, Alternaria andFusarium are antagonistic, and Alternaria was less abundant in grain with a high incidencerate of F.culmorum. Epicoccum is a strong antagonist too [25].

Species

For growth For mycotoxin production

Ref.Temperature [oC] Minimal a w Temperature [oC] Minimal a w

Range Optimum Range Range Optimum Range

Alternaria alternata 0 – 35 20 – 25 0.88 5-30 20-25 0.95-1.0 AOH

0.90 TeA

[25, 26,

28]

Fusarium culmorum <0 – 31 21 0.89 11-30 25-26 Nd [25]

Fusarium graminearum Nd 24 – 26 0.89 Nd 24-26 Nd [25]

Fusarium sporotrichoides -2 – 35 22 – 28 0.88 6-20 Nd Nd [25]

Penicillium verrucosum 0-31 20 0.81-0.83 4-31 20 0.86 [28, 29]

Penicillium expansum -6 – 35 25 – 26 0.82 – 0.85 0-31 25 0.95 [25]

Aspergillus ochraceus 8-37 24-30 0.76-0.83 12-37 25-31 0.85 OTA

0.88 PA

[28, 29]

Aspergillus parasiticus 10-43 32-33 0.84 12-40 25-30 0.87 [28, 29]

Aspergillus flavus 6 – 45 35 – 37 0.78 12-40 30 0.82 [25]

Aspergillus versicolor 4 – 39 25 – 30 0.75 15-30 23 – 29 "/>0.76 [25, 30]

OTA – ochratoxin A; PA – penicillic acid AOH – alternariol, TeA – tenuazonic acid, ND – no data

Table 2. Environmental requirements for growth and mycotoxin production


191

At 30oC, the ochratoxin production by Aspergillus ochraceus was inhibited by A.candidus,A.flavus, and A.niger in 0.995 aw. At 18oC and 0.995 aw, the interaction between Aspergillusochraceus and Alternaria alternata resulted in a significant stimulation of ochratoxin A pro‐duction [36]. Therefore, several microorganisms were reported as effective biocontrol agentsagainst several fungal plant pathogens [37]. It was determined that Trichoderma harzianumproduces a lytic enzyme, chitinase, which manifests antifungal activity against a wide rangeof fungal strains. It was found that non-toxigenic T.harzianum isolates significantly reducethe production of six types of A trichothecenes in cereals [38].

According to [39], soybean is not a favourable medium for ZEA production since it possess‐es some features that limit the production of this toxin by Fusarium isolates. Similarly, theproduction of aflatoxin B1 by Aspergillus flavus was suppressed by soybean phytoalexin –glyceollin [40].

3. Main mycotoxins

The worldwide contamination of foods and feeds with mycotoxins is a significant problem.It was estimated that 25% of the world’s crops may be contaminated with these metabolites.Mycotoxigenic fungi involved with the human food chain belong mainly to three genera As‐pergillus, Penicillium and Fusarium. The toxins produced by Alternaria have recently been ofparticular interest. The biochemistry, physiology and genetics of mycotoxigenic fungi havebeen discussed in several review articles [28, 41, 42].

Mycotoxins diffuse into grain and can be found in all grind fractions and, due to their ther‐mo-resistant properties, also in products subjected to thermal processing [43].

The characteristics of major toxins that contaminate foods and feeds in the EU, describedfrom the economic and toxicological point of view, are presented below.

3.1. Aflatoxins (AFs)

Aflatoxins are difuranocumarin derivatives. The main naturally produced aflatoxins based ontheir natural fluorescence (blue or green) are called B1, B2, G1, and G2. Aflatoxin M1 is a monohy‐droxylated derivative of AFB1 which is formed and excreted in the milk of lactating animals.AFs are very slightly soluble in water (10–30 μg/mL); insoluble in non-polar solvents; freelysoluble in moderately polar organic solvents (e.g. chloroform and methanol) and extremelysoluble in dimethyl sulfoxide. They are unstable under the influence of ultraviolet light in thepresence of oxygen, to extremes of pH (< 3, > 10) and to oxidizing agents [44].

Aflatoxins are produced only by a closely related group of aspergilli: Aspergillus flavus,A.parasiticus, and A.nomius strains [45]. These species are very widespread in the tropicaland subtropical regions of the world. Other species such as A.bombycis, A.ochraceoroseus, andA.pseudotamari are also aflatoxin-producing species, but they are found less frequently [46,47]. Aflatoxins constitute a problem concerning many commodities (nuts, spices), however,in terms of grain they are primarily problematic in case of maize. This is because only maize


can be colonised by A.flavus and related species in the field. Out of the other grains, rice is animportant dietary source of aflatoxins in tropical and subtropical areas. In regions withmoderate climate, the problem is connected with imported commodities or the local cropsthat are wet or stored in improper conditions [45]. The carcinogenicity, mutagenicity andacute toxicology of AFB1 have been well documented. The IARC determined it to be a hu‐man carcinogen (group 1A).

3.2. Ochratoxin A (OTA)

Ochratoxin A is a chlorinated isocumarin derivative, which contains a chlorinated isocoumar‐in moiety linked through a carboxyl group to L-phenylalanine via an amide bond. It is colour‐less, crystalline, and soluble in polar organic solvents compounds. This toxin is more stable inthe environment than AFs. The studies of [45] reported that thermal destruction of OTA oc‐curs after exceeding 250oC. OTA is produced by Penicillium species such as P.verrucosum, P.au‐riantiogriseum, P.nordicum, P.palitans, P. commune, P.variabile and by Aspergillus species e.g.A.ochraceus, A.melleus, A.ostanius, as well as the aspergilli species of section Nigri. In moderateclimates, the main producers of OTA are Penicillium species, while Aspergillus species domi‐nate in tropical and subtropical climates. Ochratoxin A is often found with citrinin producedby Penicillium aurantiogriseum, P.citrinum, and P.expansum [48]. Significant human exposurecomes from the consumption of grape juice, wine, coffee, spices, dried fruits and cereal-basedproducts, e.g. whole-grain breads, and in addition to this from products of animal origin, e.g.pork and pig blood-based products. The Scientific Panel on Contaminants in the Food Chain ofthe European Food Safety Authority (EFSA) has derived an OTA tolerable weekly intake(TWI) on the level of 120ng/kg b.w. The IARC [49] determined it to be a possible human carci‐nogen (group 2B). Ochratoxins are the cause of urinary tract cancers and kidney damage. In ru‐minants, ochratoxin A is divided to non-toxic ochratoxin alfa and phenylalanine [44].

3.3. Citrinin

Citrinin is a polyketide nephrotoxin produced by several species of the genera Aspergillus,Penicillium and Monascus. Some of the citrinin-producing fungi are also able to produceochratoxin A or patulin. Citrinin is insoluble in cold water, but soluble in aqueous sodiumhydroxide, sodium carbonate, or sodium acetate; in methanol, acetonitrile, ethanol, andmost other polar organic solvents. Thermal decomposition of citrinin occurs at >175 °C un‐der dry conditions, and at > 100 °C in the presence of water. The known decompositionproducts include citrinin H2 which did not show significant cytotoxicity, whereas the de‐composition product citrinin H1 showed an increase in cytotoxicity as compared to the pa‐rent compound [50].The most commonly contaminated commodities are barley, oats, andcorn, but contamination can also occur in case of other products of plant origin e.g. beans,fruits, fruit and vegetable juices, herbs and spices, and also in spoiled dairy products [50].

3.4. Fumonisins (Fs)

Fumonisins are a group of diester compounds with different tricarboxylic acids and polyhy‐dric alcohols and primary amine moiety. There are several fumonisins, but only fumonisins


193

B1 (FB1) and B2 (FB2) have been found in significant amounts. Some technological processeshydrolyze the tricarboxylic acid chain in fumonisin B1. The product of this reaction is moretoxic than fumonisin [51].

FB1 is produced by fungi from Fusarium genera, especially by F.moniliforme and F.prolifera‐tum. The study of [11] suggests that the risk of contamination with Fusarium toxins is higherfor maize and wheat than for soybean and pea. High concentrations of fumonisins are asso‐ciated with hot and dry weather, followed by the periods of high humidity. Studies on fu‐monisin residues in milk, meat and eggs are incomplete [52, 53]. Human exposureassessments on fumonisin B1 have rarely been reported. The mean daily intake in Switzer‐land is estimated to be 0.03 μg/kg bw/day. In the Netherlands the exposure estimates rangedfrom 0.006 to 7.1 μg/kg bw/day. In South Africa, the estimates ranged from 14 to 440 μg/kgbw/day, showing that the exposure to FB1 is considerably higher than in the other countriesin which exposure assessments were performed [54]. It was concluded that for Fs there wasinadequate evidence in humans for carcinogenicity. Therefore, the IARC classified Fusariummonilliforme toxins, including fumonisins, as potential carcinogens to humans (group 2B).

3.5. Zearalenone

Zearalenone is a macrocyclic lactone with high binding affinity to oestrogen receptors. ZEAis produced mainly by Fusarium graminearum and F.sporotrichoides in the field and duringstorage of commodities such as maize, barley, sorghum, and soybean. The IARC has evalu‐ated the carcinogenicity of zearalenone and found it to be a possible human carcinogen(group 2B). Residues of zearalenone in meat, milk and eggs do not appear to be a practicalproblem [53, 54].

3.6. Trichotecenes

Trichothecenes constitute a group of 50 mycotoxins produced by Fusarium, Cephalosporiumand Stachybotrys genera in different commodities. There are including T-2 toxins, deoxyniva‐lenol, nivalenol, and diacetoxyscirpenol. Beside trochothecenes, deoxynivalenol (DON, wo‐mitoxin) is probably the most widely distributed in cereal and soybean foods and feeds. Incontaminated cereals, DON derivatives such as 3-acetyl DON and 15-acetyl DON can occurin significant amounts (10 – 20%) with DON. DON is produced by closely related Fusariumgraminearum, F.culmorum and F.crokwellense species [55].

T-2 toxin produced mainly by F.sporotrichoides and F.poae is primarily associated with mouldmillet, wheat, rye, oats, and buckwheat. This toxin can be transmitted from dairy cattle feedto milk [56].

3.7. Alternaria toxins

Alternaria species, besides Fusarium, is the most isolated fungi from soybean and other cere‐als. Several species are known producers of toxic metabolites called Alternaria mycotoxins.The most important Alternaria mycotoxins include alternariol (AOH), alternariol monometh‐yl ether (AME), altertoxins I, II, and III (ATX-I, -II, III), tenuazonic acid (TeA), and altenuene


(ALT). They belong to three structural classes: dibenzopyrone derivatives, perylene deriva‐tives, and tetramic acid derivatives. Alternariol and related metabolites (AME and ALT) areproduced by Alternaria alternate, A.brassicae, A.citri, A.cucumerina, A.dauci, A.kikuchiana, A.sol‐ani, A.tenuissima, and A.tomato. These strains are known as plant, especially fruit and vegeta‐ble pathogens. In cereals, soybean and oilseeds, AOH, AME and ALT are produced mainlyby Alternaria alternata, A.tennuisima, and A.infectoria. AOH has been reported to possess cyto‐toxic, genotoxic, mutagenic, carcinogenic, and oestrogenic properties [27]. Tenuazonic acid(TeA) is a mycotoxin and phytotoxin produced primarily by Alternaria alternata and otherphytopatogenic Alternaria species. The overview of the chemical characterisation, producers,toxicity, analysis and occurrence in foodstuffs was summarised by [27].

3.8. Sterigmatocystin

Sterigmatocystin (STC) is a precursor of the aflatoxins produced mainly by many Aspergillusspecies such as A.versicolor, A.chevalieri, A.ruber, A.aureolatus, A.quadrilineatus, A.sydowi, Euro‐tium amstelodami, and less often by Penicillium, Bipolaris, Chaetomium, and Emericella genera[30]. Sterigmatocystin was reported as a fungal metabolite in mouldy wheat, rice, barley, ra‐peseed, peanut, corn, and cheeses or salami. The STC producers, occurrence and toxic prop‐erties were reviewed by [30, 57].

4. Contamination level in cereal and soybean-based food and feedproducts

Food security strategy in the European Union (EU) includes the Rapid Alert System forFood and Feed. The RASFF was established by the European Parliament and Council Regu‐lation No. 178/2002 laying down the general principles and requirements of food law, estab‐lishing the European Food Safety Authority and specifying the procedures in mattersconcerning food safety [58].

In 2002 – 2011, the number of notifications to the RASFF system due to mycotoxin contami‐nation of food was respectively: 302, 803, 880, 996, 878, 760, 933, 669, 688, 631 notificationsidentifying the presence of aflatoxin B1 (AFB1) and the amount of AFB1, B2, G1, G2, AFM1,ochratoxin A (OTA), fumonisins B1 and B2 (FB1, FB2), patulin, deoxynivalenol (DON) andzearalenone (ZEA) in such groups of foods, as nuts and milk, oilseeds, cereal, dried fruit,fruit, cocoa, coffee, herbs and spices, wine, milk, products for children. Approximately 95%of the notifications concerned foodstuffs contaminated with aflatoxins. During this period,the number of notifications regarding mycotoxin contamination of grains did not exceed15% of the total number of notifications. The data in Figure 1 show that in 2002-2011 aflatox‐ins, ochratoxin A and fumonisins were the main contaminants isolated from cereals [59].

In the research of [60], ninety-fife cereal samples from retail shops and local markets of dif‐ferent locations in Pakistan were examined in terms of the presence of aflatoxins. The resultsshowed the percentage of aflatoxin contamination samples in the commodities such as in:


195

rice (25%), broken rice (15%), wheat (20%), maize (40%), barley (20%) and sorghum (30%),while in soybean (15%). The highest contamination levels of aflatoxins were found in onewheat sample (15.5 ppb), one maize sample (13.0 ppb) and one barley sample (12.6 μg/kg).In the research of [61], seventeen samples of wheat grain from Morocco were tested for OTAand DON contamination. The results show that only two samples (11.76%) out of 17 werecontaminated with OTA, at the mean concentration of 29.4 ppb. However, seven samples(41.17%) were contaminated with DON at the mean concentration of 65.9 ppb.

The aim of our own research [15] was mycotoxic analysis of grains included in the standardmixtures used in feed formulations. Eighteen samples were tested containing seeds evenlydivided into three types: barley, wheat and corn. The tested seeds were from randomly se‐lected Polish mills: the central, western, eastern and south ones (Figure 2). The aflatoxinscontent in 51% of the screened barley samples and in 34% of the screened wheat and maizesamples did not exceed the limit set in the European Union Regulation, i.e. 4 ppb [62]. Inreference to the grain origin, it was established that grains from the central and westernparts of Poland exhibited the highest extent of AFs contamination. To compare, the AFs levelin wheat grains from various regions of Turkey was very low, ranging from 10.4 to 634.5

Mycotoxin Produced species Commodities

Aflatoxins Aspergillus flavus, A.parasiticus, A.nomius, A.bombycis,

A.ochraceoroseus, A.pseudotamari

Nuts, spices,

Cereals, maize, soybean, rice

Ochratoxin A Penicillium verrucosum, P.auriantiogriseum,

P.nordicum, P.palitans, P.commune, P.variabile,

Aspergillus ochraceus, A.melleus, A.niger,

A.carbonarius, A.sclerotiorum, A.sulphureus

Cereals, fruits, spices, coffee,

Food of animal origin

Citrinin Penicillium citrinum, P.verrucosum, P.viridicatum,

Monascus purpureus

Oats, rice, corn, beans, fruits, fruit and

vegetable juices, herbs and spices

Sterigmatocystin Aspergillus versicolor, A.nidulans, A.chevalieri, A.ruber,

A.aureolatus, A.quadrilineatus, Eurotium amstelodami

Cereals, cheese

Zearalenone Fusarium graminearum, F.sporotrichoides, F.culmorum,

F.cerealis, F.equiseti, F.incarnatum

Maize, soybean, cereals

Deoksynivalenol Fusarium graminearum, F.culmorum, F.crokwellense Maize, soybean, cereals

Fumonisins Fusarium proliferatum, F.verticillioides, Maize, soybean, cereals

Alternariol, alternariol

monomethyl ether

Alternaria alternata, A.brassicae, A.capsici-anui, A.citri,

A.cucumerina, A.dauci, A.kikuchiana, A.solani,

A.tenuissima, A.tomato, A.longipes, A.infectoria,

A.oregonensis

Vegetables, fruit, cereals, soybean

Tenuazonic acid Alternaria alternata, A.capsici-anui, A.citri, A.japonica,

A.kikuchiana, A.mali, A.solani, A.oryzae, A.porri,

A.radicina, A.tenuissima, A.tomato, A.longipes

Vegetables, fruit, cereals, soybean

Table 3. Mycotoxigenic fungi and mycotoxins


ng/kg [63], whereas in the samples of barley, wheat, and oat grains from Sweden it was con‐tained between 50 and 400 ppb [64].

notifications regarding mycotoxin contamination of grains did not exceed 15% of the total number of notifications. The data in

Figure 1 show that in 2002-2011 aflatoxins, ochratoxin A and fumonisins were the main contaminants isolated from cereals [59].

Figure 1. The number of notifications received by RASFF on mycotoxins in cereals in 2002-2011

In the research of [60], ninety-fife cereal samples from retail shops and local markets of different locations in Pakistan were

examined in terms of the presence of aflatoxins. The results showed the percentage of aflatoxin contamination samples in the

commodities such as in: rice (25%), broken rice (15%), wheat (20%), maize (40%), barley (20%) and sorghum (30%), while in

soybean (15%). The highest contamination levels of aflatoxins were found in one wheat sample (15.5 ppb), one maize sample (13.0

ppb) and one barley sample (12.6 μg/kg). In the research of [61], seventeen samples of wheat grain from Morocco were tested for

OTA and DON contamination. The results show that only two samples (11.76%) out of 17 were contaminated with OTA, at the

mean concentration of 29.4 ppb. However, seven samples (41.17%) were contaminated with DON at the mean concentration of 65.9

ppb.

The aim of our own research [15] was mycotoxic analysis of grains included in the standard mixtures used in feed formulations.

Eighteen samples were tested containing seeds evenly divided into three types: barley, wheat and corn. The tested seeds were from

randomly selected Polish mills: the central, western, eastern and south ones (Figure 2). The aflatoxins content in 51% of the

screened barley samples and in 34% of the screened wheat and maize samples did not exceed the limit set in the European Union

Regulation, i.e. 4 ppb [62]. In reference to the grain origin, it was established that grains from the central and western parts of

Poland exhibited the highest extent of AFs contamination. To compare, the AFs level in wheat grains from various regions of

Turkey was very low, ranging from 10.4 to 634.5 ng/kg [63], whereas in the samples of barley, wheat, and oat grains from Sweden it

was contained between 50 and 400 ppb [64].

Figure 2. Level of contamination with aflatoxins in grains coming from different regions of Poland

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2002 2003 2004 2005 2006 2007 2008 2009 2010 2011

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aflatoxins deoxynivalenol fumonisins ochratoxin A zearalenone

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Central Poland Western Poland Eastern Poland Polish Noon

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notifications regarding mycotoxin contamination of grains did not exceed 15% of the total number of notifications. The data in

Figure 1 show that in 2002-2011 aflatoxins, ochratoxin A and fumonisins were the main contaminants isolated from cereals [59].


In the research of [60], ninety-fife cereal samples from retail shops and local markets of different locations in Pakistan were

examined in terms of the presence of aflatoxins. The results showed the percentage of aflatoxin contamination samples in the

commodities such as in: rice (25%), broken rice (15%), wheat (20%), maize (40%), barley (20%) and sorghum (30%), while in

soybean (15%). The highest contamination levels of aflatoxins were found in one wheat sample (15.5 ppb), one maize sample (13.0

ppb) and one barley sample (12.6 μg/kg). In the research of [61], seventeen samples of wheat grain from Morocco were tested for

OTA and DON contamination. The results show that only two samples (11.76%) out of 17 were contaminated with OTA, at the

mean concentration of 29.4 ppb. However, seven samples (41.17%) were contaminated with DON at the mean concentration of 65.9

ppb.

The aim of our own research [15] was mycotoxic analysis of grains included in the standard mixtures used in feed formulations.

Eighteen samples were tested containing seeds evenly divided into three types: barley, wheat and corn. The tested seeds were from

randomly selected Polish mills: the central, western, eastern and south ones (Figure 2). The aflatoxins content in 51% of the

screened barley samples and in 34% of the screened wheat and maize samples did not exceed the limit set in the European Union

Regulation, i.e. 4 ppb [62]. In reference to the grain origin, it was established that grains from the central and western parts of

Poland exhibited the highest extent of AFs contamination. To compare, the AFs level in wheat grains from various regions of

Turkey was very low, ranging from 10.4 to 634.5 ng/kg [63], whereas in the samples of barley, wheat, and oat grains from Sweden it

was contained between 50 and 400 ppb [64].


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45

50

2002 2003 2004 2005 2006 2007 2008 2009 2010 2011

year

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aflatoxins deoxynivalenol fumonisins ochratoxin A zearalenone

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barley wheat corn



197

Figure 3. Level of contamination with ochratoxina A in grains coming from different regions of Poland

The OTA level in the examined grains collected from mills in central, eastern and southern Poland was low and ranged from 0.5 to

2.5 ppb (Figure 3). Therefore, it did not exceed the permissible limit set by the European Union (Commission Regulation No.

105/2010), i.e. 5 ppb [65]. Only in barley coming from a mill located in western Poland, the OTA level exceeded the limits fivefold.

The extent of OTA contamination of barley, wheat, and maize grain from various regions of Mexico was also low and recorded 0.17

ppb, 0.42 ppb, and 1.08 ppb, respectively. Only 1 out of 20 examined maize grains showed the OTA level of 7.22 [66]. To compare,

the OTA concentration in barley and wheat grain from the UK equalled from 1 to 33 ppb [67]. In the research of [68], among others,

the levels of AFs and OTA in 532 grain and feed samples from Poland from 2002 and 2003 were determined. The average

mycotoxin concentration levels were similar and quite low, i.e. AFs - 0.3 ppb and OTA - 1.1 ppb in grains and feeds from 2002, and

respectively, AFs 3.1 and 1.0 ppb and OTA 0.5 and 0.7 OTA in samples from 2003. The authors of the study stressed that in 2002

and 2003 the harvesting seasons were hot and dry, which might have resulted in the low extent of fungi contamination of the

examined grain. Although the extent of mycotoxin contamination of grain in the quoted studies varies, their authors concur that it

is a serious issue whose scale depends on the microclimate during arable farming and the subsequent phases, i.e. grain storage. It

was reported that no mycotoxins were found in barley samples stored for 20 weeks at 15% seed humidity, whereas the samples of

wheat stored for the same period of time at 19% humidity recorded relatively high concentration levels: OTA - 24 ppb, citrinin - 38

ppb, and sterigmatocystin even up to 411 ppb [69].

The aim of our research was the assessment of cereal products available in trade and meant for direct consumption as for

contamination with selected mycotoxins. The research included corn flakes, corn flakes with nuts and honey, various kinds of

breakfast cereal products and muesli containing dried fruit, nuts as well as cereal and coconut flakes (15 samples). None of the

products was contaminated with AB1 on the level exceeding the acceptable limits (2 ppb). The presence of ochratoxin A exceeding

the amount of 3 ppb was discovered in four samples (two kinds of corn flakes, exotic muesli and traditional muesli). The

contamination with that toxin equalled 4.5 ppb on average. According to the current regulation, contamination of breakfast flakes

with deoxynivalenol DON should not exceed 500 ppb. Four samples (containing corn) exceeded this limit by 50%. In case of one

sample, DON contamination was very high, almost three times higher than the acceptable level [19].

Mycotoxin contamination of soybean is not considered a significant problem as compared to commodities such as corn, cottonseed,

peanuts, barley and other grains. In the early surveys conducted by the U.S. Department of Agriculture (USDA), 1046 soybean

samples collected from different regions of the United States were examined for aflatoxins contamination. Aflatoxin presence was

confirmed at low levels (7-14 ppb) in only two of the tested samples [70]. In the research of [71], fifty-five samples of soybean meals

were analysed for the content of aflatoxins, deoxynivalenol (DON), zearalenone (ZEA) and ochratoxin A (OTA). Regarding

aflatoxins, only AFB1 was detected in 32 out of the 51 non-suspicious samples, but the maximal concentration found was only 0.41

ppb. ZEA was detected in 23 out of the 51 samples with a maximum concentration of 18 ppb. DON could be detected only in one

suspicious sample in a low concentration of 104 ppb. OTA was found in 5 samples, with the greatest concentration being only 1

ppb.

The research of [72] tested 122 soybean samples that came from Asia and the Pacific region. Aflatoxin was found in only in 2%

(maximum of 13 ppb, median 9 ppb), zearalenone in 17% (maximum 1078 ppb, median 57 ppb), ochratoxin in 13% (maximum 11

ppb, median 7 ppb), and DON and fumonisins each in 7% of the analyzed samples (DON: maximum 1347ppb, median 264 ppb;

fumonisins: maximum 331 ppb, median 154 ppb). In maize and maize products, the levels of fumonisins varied from 0.07 to 38.5

ppm in Latin America, from 0.004 to 330 ppm in North America, from 0.02 to 8.85 ppm in Africa, and from 0.01 to 153 ppm in Asia.

The data available for Europe varied from 0.007 to 250 ppm in maize, and from 0.008 to 16 ppm in maize products. [54].

5. Influence of mycotoxins on human and animal organisms

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Figure 3. Level of contamination with ochratoxina A in grains coming from different regions of Poland

The OTA level in the examined grains collected from mills in central, eastern and southernPoland was low and ranged from 0.5 to 2.5 ppb (Figure 3). Therefore, it did not exceed thepermissible limit set by the European Union (Commission Regulation No. 105/2010), i.e. 5ppb [65]. Only in barley coming from a mill located in western Poland, the OTA level ex‐ceeded the limits fivefold. The extent of OTA contamination of barley, wheat, and maizegrain from various regions of Mexico was also low and recorded 0.17 ppb, 0.42 ppb, and1.08 ppb, respectively. Only 1 out of 20 examined maize grains showed the OTA level of 7.22[66]. To compare, the OTA concentration in barley and wheat grain from the UK equalledfrom 1 to 33 ppb [67]. In the research of [68], among others, the levels of AFs and OTA in 532grain and feed samples from Poland from 2002 and 2003 were determined. The average my‐cotoxin concentration levels were similar and quite low, i.e. AFs - 0.3 ppb and OTA - 1.1 ppbin grains and feeds from 2002, and respectively, AFs 3.1 and 1.0 ppb and OTA 0.5 and 0.7OTA in samples from 2003. The authors of the study stressed that in 2002 and 2003 the har‐vesting seasons were hot and dry, which might have resulted in the low extent of fungi con‐tamination of the examined grain. Although the extent of mycotoxin contamination of grainin the quoted studies varies, their authors concur that it is a serious issue whose scale de‐pends on the microclimate during arable farming and the subsequent phases, i.e. grain stor‐age. It was reported that no mycotoxins were found in barley samples stored for 20 weeks at15% seed humidity, whereas the samples of wheat stored for the same period of time at 19%humidity recorded relatively high concentration levels: OTA - 24 ppb, citrinin - 38 ppb, andsterigmatocystin even up to 411 ppb [69].

The aim of our research was the assessment of cereal products available in trade and meantfor direct consumption as for contamination with selected mycotoxins. The research includ‐ed corn flakes, corn flakes with nuts and honey, various kinds of breakfast cereal products


and muesli containing dried fruit, nuts as well as cereal and coconut flakes (15 samples).None of the products was contaminated with AB1 on the level exceeding the acceptable lim‐its (2 ppb). The presence of ochratoxin A exceeding the amount of 3 ppb was discovered infour samples (two kinds of corn flakes, exotic muesli and traditional muesli). The contami‐nation with that toxin equalled 4.5 ppb on average. According to the current regulation, con‐tamination of breakfast flakes with deoxynivalenol DON should not exceed 500 ppb. Foursamples (containing corn) exceeded this limit by 50%. In case of one sample, DON contami‐nation was very high, almost three times higher than the acceptable level [19].

Mycotoxin contamination of soybean is not considered a significant problem as compared tocommodities such as corn, cottonseed, peanuts, barley and other grains. In the early surveysconducted by the U.S. Department of Agriculture (USDA), 1046 soybean samples collectedfrom different regions of the United States were examined for aflatoxins contamination.Aflatoxin presence was confirmed at low levels (7-14 ppb) in only two of the tested samples[70]. In the research of [71], fifty-five samples of soybean meals were analysed for the con‐tent of aflatoxins, deoxynivalenol (DON), zearalenone (ZEA) and ochratoxin A (OTA). Re‐garding aflatoxins, only AFB1 was detected in 32 out of the 51 non-suspicious samples, butthe maximal concentration found was only 0.41 ppb. ZEA was detected in 23 out of the 51samples with a maximum concentration of 18 ppb. DON could be detected only in one sus‐picious sample in a low concentration of 104 ppb. OTA was found in 5 samples, with thegreatest concentration being only 1 ppb.

The research of [72] tested 122 soybean samples that came from Asia and the Pacific region.Aflatoxin was found in only in 2% (maximum of 13 ppb, median 9 ppb), zearalenone in 17%(maximum 1078 ppb, median 57 ppb), ochratoxin in 13% (maximum 11 ppb, median 7 ppb),and DON and fumonisins each in 7% of the analyzed samples (DON: maximum 1347ppb,median 264 ppb; fumonisins: maximum 331 ppb, median 154 ppb). In maize and maizeproducts, the levels of fumonisins varied from 0.07 to 38.5 ppm in Latin America, from 0.004to 330 ppm in North America, from 0.02 to 8.85 ppm in Africa, and from 0.01 to 153 ppm inAsia. The data available for Europe varied from 0.007 to 250 ppm in maize, and from 0.008to 16 ppm in maize products. [54].

5. Influence of mycotoxins on human and animal organisms

Effects of mycotoxins on human and animal health are now increasingly recognised. Myco‐toxins enter human and animal dietary systems mainly through ingestion, but increasingevidence also points to inhalation as another entry route. Mycotoxins exhibit a wide array ofbiological effects and individual mycotoxins can be [73]:

• carcinogenic - aflatoxins, ochratoxins, fumonisins, and possibly patulin;

• mutagenic - aflatoxins and sterigmatocystin;

• hematopoietic - aflatoxins and trichothecenes. Hemotopoiesis refers to the production ofall types of blood cells from the primitive cells stem cells in the bone marrow. The dys‐


199

function of hematopoiesis leads firstly to the decrease in the number of neutrophils, thusperturbing the animal’s immune system and subsequently to the decrease in red bloodcells, which leads to anemia;

• hepatotoxic - aflatoxins, ochratoxins, fumonisins. All of them induce significant liverdamage when given to animals;

• nephrotoxigenic - ochratoxins, citrinin, trichothecenes, and fumonisins;

• teratogenic - aflatoxin B1, ochratoxin A, T-2 toxin, sterigmatocystin, and zearalenone;

• oestrogenic - zearalenone;

• neurotoxic - ergot alkaloids, fumonisins, deoksynivalenol. The effects of mycotoxins arebest evidenced by vomiting and taste aversion produced by DON, seizures, focal malataand liquefaction of the brain tissue, possibly mediated by sphingolipid synthesis underthe influence of fumonisins, staggering and trembling produced by many tremorgenicpenitrem mycotoxins seizures and other neural effects of ergot alkaloids and parasympa‐thomimetic activity resulting from the effects of the metabolite slaframine for selected re‐ceptors in the nervous system

• immunosupresive - several mycotoxins. The predominant mycotoxins in this regard areaflatoxins, trichothecenes, and ochratoxin A. However, several other mycotoxins such asfumonisins, zearalenone, patulin, citrinin, and fescue and ergot alkaloids have beenshown to produce some effects on the immune system.

Table 4 presents the groups of mycotoxins which are most harmful to human and animalorganisms, together with the chosen disease symptoms they cause.

5.1. Negative effects of mycotoxins on humans

Mycotoxicoses can be divided into acute and chronic. Acute toxicity usually has a rapid on‐set and obvious toxic response, chronic exposure is characterized by chronic doses over along period of time and may lead to cancer and other effects that are generally irreversible.The symptoms of mycotoxicosis depend on the type, amount and duration of exposure, age,health and sex of the exposed individual, and many poorly understood synergistic effectsinvolving genetics, dietary status, and interaction with other toxic contaminants. Thus, theseverity of mycotoxin poisoning can be compounded by factors such as vitamin deficiency,caloric deprivation, alcohol abuse, and infectious disease status. Mycotoxicosis is difficult todiagnose because doctors do not have experience with this disease and its symptoms are sowide that it mimics many other conditions [74, 75].

Aflatoxicosis is toxic hepatitis leading to jaundice and, in severe cases, death. AFB1 has beenextensively linked to human primary liver cancer and was classified by the InternationalAgency for Research on Cancer (IARC) as a human carcinogen (Group 1A - carcinogens)[49]. Although acute aflatoxicosis in humans is rare, several outbreaks have been reported.In 2004, one of the largest aflatoxicosis outbreaks in Kenya, resulting in 317 cases and 125deaths was observed. Contaminated corn was responsible for the outbreak, and officials


found the level of aflatoxin B1 as high as 4400 ppb [76]. Research in Gambian children andadults reported a strong association between aflatoxin exposure and impaired immunocom‐petence suggesting that the consumption of aflatoxin reduces resistance to infections in hu‐man populations [77, 78]. In 1974, an epidemic of hepatitis in India affected 400 peopleresulting in 100 deaths. The death was due to consumption of corn that was contaminatedwith A. flavus containing up to 15000 ppb of aflatoxins [79].

Ochratoxin A was the cause of epithelial tumours of the upper urinary tract in the Balkans[80, 81]. The condition is known as Balkan endemic nephropathy. Despite the seriousness ofthe problem, the study did not explain the mechanism of action and the size of OTA carcino‐genicity in humans [82]. Ochratoxin has been detected in blood in 6-18% of the human pop‐ulation in some areas where Balkan endemic nephropathy is prevalent. Ochratoxin A hasalso been found in human blood samples from outside the Balkan Peninsula. In some sur‐vey, over 50% of the tested samples were contaminated. A highly significant correlation wasobserved between Balkan nephropathy and urinary tract cancers, particularly tumours ofthe renal pelvis and ureter. However, no data have been published that establishes a directcausal role of ochratoxin A in the etiology of these tumours [81].

Mycotoxin Toxicity class according to

International Agency for Research

on Cancer (IARC)

Symptoms and diseases

Aflatoxins I * aflatoxicosis, primary liver cancer, lung neoplasm, lung

cancer, failure of the immune system, vomiting, depression,

hepatitis, anorexia, jaundice, vascular coagulation

Ochratoxins II B ** renal diseases, nephropathy, anorexia, vomiting, intestinal

haemorrhage, tonsillitis, dehydration

Fumonisins II B ** diseases of the nervous system, cerebral softening,

pulmonary oedema, liver cancers, kidney diseases,

oesophagus cancers, anorexia, depression, ataxia, blindness,

hysteria, vomiting,

hypotension

Zearalenone - reproduction disruptions, abortions, pathological changes in

the reproductive system

Trichothecenes - nausea, vomiting, haemorrhages, anorexia, alimentary toxic

aleukia, failure of the immune

system, infants’ lung bleeding, increased thirst, skin rash

*The agent (mixture) is carcinogenic to humans. The exposure circumstance entails exposures that are carcinogenic tohumans

**The agent (mixture) is possibly carcinogenic to humans. The exposure circumstance entails exposures that are possi‐bly carcinogenic to humans.

Table 4. The list of adverse effects of the chosen mycotoxins.


201

Fumonisin B1 was classified by the IARC as a group 2B carcinogen (possibly carcinogenic forhumans) [44]. Fumonisins, which inhibit the absorption of folic acid through the foliate re‐ceptor, have also been implicated in the high incidence of neural tube defects in the ruralpopulation known to consume contaminated corn, such as the former Transkei region ofSouth Africa and some areas of Northern China [75, 83].

Trichothecenes have been proposed as potential biological warfare agents. In the years1975-1981, T-2 toxin was implicated as a chemical agent "yellow rain" used against the LaoPeoples Democratic Republic. A study conducted from 1978 to 1981 in Cambodia revealedthe presence of T-2 toxin, DON, ZEA, and nivalenol in water and leaf samples taken fromthe affected areas [75, 84]. Clinical symptoms proceeding to death included vomiting, diar‐rhoea, bleeding, and difficulty with breathing, pain, blisters, headache, fatigue and dizzi‐ness. There also occurred necrosis of the mucosa of the stomach as well as the smallintestine, lungs and liver [85]. One disease outbreak was recorded in China and was associ‐ated with the consumption of scabby wheat containing 1000-40000 ppb of DON. The diseaseis characterized by gastrointestinal symptoms. Also, in India there took place a reported in‐fection associated with the consumption of bread made from contaminated wheat (DON350-8300 ppb, acetyldeoxynivalenol 640-2490 ppb, NIV 30-100 ppb and T-2 toxin 500-800ppb). The disease is characterized by gastrointestinal symptoms and throat irritation, whichdeveloped within 15 minutes to one hour after ingestion of the contaminated bread [81].

5.2. Negative effects of mycotoxins on animal

Animals may show varied symptoms upon contact with mycotoxins, depending on the ge‐netic factors (species, breed, and strain), physiological factors (age, nutrition) and environ‐mental factors (climatic conditions, rearing and management). The natural contaminationwith mycotoxins in animal feed usually does not occur at the levels that may cause acute orovert mycotoxicosis, such as hepatitis, bleeding, nephritis and necrosis of the oral and enter‐ic epithelium, and even death. It is often difficult to observe and diagnose the symptoms ofthe disease, but it certainly is the most common form of mycotoxicosis in farm animals, af‐fecting such parameters as productivity, growth and reproductive performance, feed effi‐ciency, milk and egg production.

The negative effects of mycotoxins on the performance of poultry have been shown in nu‐merous studies. For example, feeding the broilers with feed containing an AFs mixture (79%AFB1, 16% AFG1, AFB2 4% and 1% AFG2) in the concentration of 3.5 ppm decreased theirbody weight and increased their liver and kidney weight [75, 86]. Feeding OTA (0.3-1 ppm)to broilers reduced glycogenolysis and dose-dependent accumulation of glycogen in the liv‐er. These negative metabolic reactions were attributed to inhibition of cyclic adenosine 3',5'-monophosphate-dependent protein kinase, and were reflected in reduced efficiency of feedutilization and teratogenic malformations [75].

Fusarium mycotoxins proved to be harmful to poultry. In addition to reduced feed intakeand weight gain, sore mouth, cheeks and plaque formation was observed after 7-day-oldchicks were exposed to T-2 toxin (4 or 16 ppm) [75, 87]. Pigs are among the most sensitivespecies to mycotoxins. In the study by [88], pigs in response to AFs (2 ppm), OTA (2 ppm),


or both were evaluated. Compared to the control group, the body weight gains were re‐duced by 26, 24 and 52% for animals consuming diets containing AFs, OTA, or both, respec‐tively. Additional symptoms in pig ochratoxicosis were anorexia, fainting, uncoordinatedmovements, and increased water consumption and urination. Pigs also are susceptible toother mycotoxins, such as fumonsins and ergot alkaloids. Fumonisin B1, for example, hasbeen shown to cause pulmonary oedema and heart and respiratory dysfunction. The symp‐toms of swine pulmonary oedema included dyspnoea, cyanosis, and death [89, 90]. Myco‐toxic porcine nephropathy is a serious disease, often associated with pigs consuming feedcontaminated with OTA, especially in Scandinavian region. In addition to the enlarged andpale kidneys (with vascular lesions and white spots), morphological changes include a prox‐imal tubular injury, epithelial atrophy, fibrosis and hyalinization of renal glomerular [80,81]. Negative effects of ZEA on pigs’ reproductive function have also been demonstrated[91]. Oestrogenic effects of ZEA on gilts and sows include oedematous uterus and ovariancysts, increased maturation of follicles, more numerous litters or decreased fertility [92].

Aflatoxins affect the quality of the milk produced by dairy cows and result in a carry-over ofAFM1 with AFB1-contaminated feed. Ten ruminally-canulated Holstein cows received AFB1

(13 mg per cow daily) through a hole in the rumen for 7 days. The AFM1 levels in the milk ofthe treated cows ranged from 1.05 to 10.58 ng/L. The carry-over rate was higher in early lac‐tation (2-4 weeks) compared to late lactation (34 -36 weeks) [75, 93]. The T-2 toxin causes ne‐crosis of the lymphoid tissues. Bovine infertility and natural abortion in the last trimester ofpregnancy also result from consumption of feed contaminated with T-2 toxin. Calves con‐suming T-2 toxin in the amount of 10-50 mg/kg of feed showed abomasal ulcers and slough‐ing of papillae in the rumen [75, 94, 95].

6. Current EU regulations concerning mycotoxins

Since the discovery of aflatoxins in the 1960s, regulations have been established in manycountries to protect consumers from harmful mycotoxins that can contaminate foods. Maxi‐mum levels of mycotoxins have been established by the European Commission after consul‐tations with the Scientific Committee for Food, based on the analysis of scientific datacollected by EFSA and the Codex Alimentarius.

These data include [73, 96]:

• toxicological properties of mycotoxins,

• mycotoxin dietary exposure,

• distribution of concentrations of mycotoxins in raw materials or a product batch

• availability of analytical methods,

• regulations in other countries with which trade contacts exist.

The first two factors provide the information necessary for risk assessment and exposure as‐sessment, respectively. Risk assessment is the scientific evaluation of the likelihood of


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known or potential adverse health effects resulting from human exposure to food-bornehazards. It is a fundamental scientific basis for the notification of regulations. The third andfourth factors are important factors in enabling the practical enforcement of mycotoxins,through appropriate procedures as regards sampling and analysis. The last factor is the onlyone economic in nature, but it is equally important in decision-making to establish reasona‐ble rules and restrictions for mycotoxins in foods and feeds [96].

According to the Commission Regulations, the maximum levels should be set at a strict lev‐el, which is reasonably achievable by following good agricultural and manufacturing practi‐ces and taking into account the risk related to the consumption of food. Health protection ofinfants and young children requires establishing the lowest maximum levels, which is ach‐ievable through the selection of raw materials used for the manufacturing of foods for thisvulnerable group of consumers. Development of international trade, progress in research fo‐cused on mycotoxin food contamination and their toxicological properties cause changes inthe mycotoxin-related legislationacross the European Union. The Commission Regulation466/2001 [97] setting the maximum levels for certain contaminants in foodstuffs has beensubstantially amended many times. Te current maximum levels for mycotoxins in food arespecified by the Commission Regulation EU 1881/2006 and the Commission Regulation EU105/2010 as regards OTA, the Commission Regulation EU 165/2010 as regards aflatoxins,and the Commission Regulation EU 1126/2007 as regards Fusarium toxins [62, 65, 98, 99].There have also been established maximum levels for aflatoxins, ochratoxin A, patulin, andFusarium toxin (fumonisin, deoxynivalenol, zearalenone) in different products: nuts, cereals,dried fruit, unprocessed cereals, processed cereal-based food, coffee, wine, spices, and liquo‐rices [62, 65, 97-99].

The number of countries that have regulations concerning mycotoxins is continuously in‐creasing, and at least 100 countries are known to have founded specific limits for differentcombinations of mycotoxins and commodities, often accompanied by the prescribed or rec‐ommended procedures for sampling and analysis [100]. Specific regulations for food in dif‐ferent world regions were summarized by [101].

As for feeds, the legal situation is somewhat different and only aflatoxin B1 is regulated bythe Directive 2002/32/EC on undesirable substances in animal food amended by the Com‐mission Directive (EC) 100/2003 [102, 103]. For other mycotoxins, such as deoxynivalenol,zearalenone, ochratoxin A and fumonisin B1 and B2 - only non-binding recommendation val‐ues in the Commission Recommendation 2006/57/EC [104] are determined for feeds (Table6). This results from the fact that with the exception of aflatoxin-contaminated feed whicheither directly or indirectly affects human health, there is only a slight transfer to animalproducts [104, 105].

Table 5 presents the current maximum levels of mycotoxin content as regards cereals andcereal-based foods and feeds.

Mycotoxins in agricultural commodities are distributed heterogeneously. Therefore, sam‐pling plays a crucial role in making the estimation of the levels of mycotoxin presence moreprecise. In order to obtain representative samples, sampling procedures, and particularly


homogenisation, for different matrix types have been regulated. The EU Commission Regu‐lation (EC) 401/2006 established the methods of sampling and analysis for the official controlof mycotoxins in foodstuffs [106]. Official sampling plans for aflatoxins in dry figs, ground‐nuts, peanuts, oilseeds, apricot kernels and tree nuts and for ochratoxins in coffee and liquo‐rice root are provided in the Commission Regulation (EU) No 178/2010 [107 ]. The samplingfrequency and the method of sampling for cereals and cereal products for lots >50 tonnesand <50 tonnes, as well as for retail packed products were presented. Moreover, the proce‐dures of subdivision of lots into sublots depending on the product and lot weight were alsosummarised [106, 107].

According to the current regulations where no specific methods for the determination ofmycotoxin levels in food are required by the EU regulations, laboratories may select anymethod provided that they meet the relevant criteria presented in [106, 107]. These criteriaare different in relation to individual mycotoxins, and the limit of detection, precision, andrecovery depends on the concentration range. The analytical results must be submittedcorrected or uncorrected for recovery and the level of recovery expressed in % must be re‐ported too.

The main analytical procedures for the determination of the major mycotoxins from com‐plex biological matrices consist of the following steps: sampling, extraction, purification, de‐tection, quantification, and finally confirmation. The current development in mycotoxinestimation was reviewed by [108-110].

Regulation Matrix Maximum levels [ppb]

AFB1 OTA DON ZEA F

FOOD

CommissionRegulation (EU)165/2010

All cereals and all products derived from cereals 2.0 - - - -

Maize and rice 5.0 - - - -

Processed cereal-based foods for infants andyoung children

0.10 - - - -

CommissionRegulation (EC)1126/2007

Unprocessed cereals - - 1250 100 -

Unprocessed durum wheat and oats - - 1750 - -

Pasta (dry) - - 750 - -

Bread (including small bakery wares), pastries,biscuits, cereal snacks and breakfast cereals

- - 500 50 -

Maize-based breakfast cereals and maize-basedsnacks

- - - - 800

Unprocessed maize with the exception ofunprocessed maize intended to be processed bywet milling

- - 1750 350 4000

Cereals intended for direct humanconsumption, cereal flour, bran and germ as anend product marketed for direct humanconsumption

- - 750 75 -


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Regulation Matrix Maximum levels [ppb]

AFB1 OTA DON ZEA F

Milling fractions of maize and milling productswith particle size "/> 500 micron not used fordirect human consumption

- - 750 200 1400

Milling fractions of maize and maize millingproducts with particle size ≤ 500 micron notused for direct human consumption

- - 1250 300 2000


- - 200 20 200

Processed maize-based foods for infants andyoung children

- - - 20 -

CommissionRegulation (EC)1881/2006

Unprocessed cereals - 5.0 - - -

All products derived from unprocessed cereals,including processed cereal products and cerealsintended for direct human consumption

- 3.0 - - -


- 0.50 - - -

FEED

CommissionRecommendation(EC) 576/2006

Cereals and cereal products with the exceptionof maize by-products

- 250 8000 2000 -

Maize by-products - - 12000 3000 -

Complementary and complete feedingstuffs forpigs

- 50 900 250 -

Complementary and complete feedingstuffs forcalves, lambs and kids

- - 2000 500 -

Complementary and complete feedingstuffs forpoultry

- 100 - - -

Commission Directive(EC) 100/2003

All feed materials 20 - - - -

Complete feedingstuffs for dairy animals 5 - - - -

Complete feedingstuffs for calves and lambs 10 - - - -

Complete feedingstuffs for pigs, poultry, cattle,sheep and goats

20 - - - -

(-) limit not established; AFB1 – aflatoxin B1; OTA – ochratoxin A; ZEA – zearalenone; DON – deoxynivalenol; F – fumoni‐sins

Table 5. Legislation on mycotoxins as regards cereals and cereal-based foods and feeds

7. Prevention strategies of exposure to mycotoxins

Several codes of practice have been developed by Codex Alimentarius for the preventionand reduction of mycotoxins in cereals, peanuts, apple products, and other raw materials. Inorder for this practice to be effective, it will be necessary for the producers in each country toconsider the general principles given in the Code, taking into account their local crops, cli‐


mate, and agronomic practices, before attempting to implement the provisions specified inthe Code. The recommendations for the reduction of various mycotoxins in cereals are div‐ided into two parts: recommended practices based on Good Agricultural Practice (GAP) andGood Manufacturing Practice (GMP); a complementary management system to consider inthe future is the use of Hazard Analysis Critical Control Point (HACCP) [111].

Recommendations to be taken into account before the harvest in order to reduce the risk ofmould contamination and mycotoxin production include [112]:

• use certified seed or ensure it is free from fungal infections;

• avoid drought stress – irrigate if possible;

• sow the seed as early as possible, so that crop matures early;

• when practising minimum or zero tillage, remove crop residues;

• weed regularly;

• control insect and bird pests;

• rotate crops;

• avoid nutrient stress – apply the appropriate amount of organic or inorganic fertiliser;

• plant resistant varieties where these are available

The main mycotoxin hazards associated with pre-harvest in Europe are the toxins that areproduced by fungi belonging to the genus Fusarium in the growing crops. It is important tonote that although Fusarium infection is generally considered to be a pre-harvest problem, itis certainly possible for poor drying practices to lead to crops’ susceptibility in storage andmycotoxin contamination [113]. This part of the book will discuss some pre-harvest strat‐egies appropriate to reduce the prevalence of fungi belonging to the genus Fusarium andtheir mycotoxins.

7.1. Resistance

There are inherent differences in the susceptibility of cereal species to Fusarium infections.The differences between crop species appear to vary between countries. This is probablydue to the differences in the genetic pool within each country’s breeding program and thediverse environmental and agronomic conditions in which crops are cultivated [114, 115]. Itwas observed that oats had higher levels of DON than barley and wheat in Norway from1996 to 1999, whereas the DON levels in wheat, barley and oats were similar when grownunder the same field conditions in Western Canada in 2001 [116].

7.2. Field management

Crop rotation

Numerous studies have shown that fumonisins or DON contamination in wheat is affectedby the previous crop. It was shown that a higher incidence of Fs occurred in wheat after


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maize and, in particular, in wheat after a succession of two maize crops and in wheat fol‐lowing grain maize compared to silage maize. In Ontario, Canada, in 1983, the fields wheremaize was the previous crop had a significantly higher incidence of fumonisins than thefields where the previous crop was a small grain cereal or soybean [117]. In a repeatedstudy, the following year, the fields where maize was the previous crop had a 10-fold DONcontent than the fields following a crop other than maize [118]. The research of [119] foundhigher levels of fumonisins in wheat following wheat rather than wheat following fallow.

An observational study performed using commercial fields in Canada [120] identified signif‐icantly lower DON content in wheat following soybean or wheat, compared to wheat fol‐lowing maize. In New Zealand, an observational study determined that higher levels ofDON occurred in wheat grown after maize (mean = 600 ppb) and after grass (mean = 250ppb), compared to small grain cereals (mean = 90 ppb) and other crops (mean = 70 ppb). Thehighest levels were recorded in wheat-maize rotations [121].

Codex recommends that crops such as potatoes, other vegetables, clover and alfalfa thatare not hosts to Fusarium species should be used in rotation to reduce the inoculum inthe field [122].

7.3. Soil cultivation

Soil cultivation can be divided into ploughing, where the top 10-30 cm of soil are inverted; min‐imum tillage, where the crop debris is mixed with the top 10-20 cm of soil; and no till, whereseed is directly drilled into the previous crop stubble with minimum disturbance to the soilstructure [111]. In the 1990s, a large observational study of Fs and DON was conducted in Ger‐many (n=1600). The DON concentration of wheat crops after maize was ten-times higher in thefield that was min-tilled compared to the ploughed one [123]. In wheat the DON concentra‐tion after min-till was 1300 ppb, after no-till it was 700 ppb and after ploughing it was 500 ppb[120]. Studies in France have determined that crop debris management can have a large im‐pact on the DON concentration at harvest, particularly after maize. The highest DON concen‐tration was found after no-till, followed by min-till, whereas the lowest DON levels wererecorded after ploughing. The reduction in DON has been linked to the reduction in crop resi‐due on the soil surface [124]. Large replicated field trials in Germany identified that there was asignificant interaction between the previous crop and the cultivation technique [125]. Follow‐ing sugar beet, there was no significant difference in the DON concentration between wheatplots receiving different methods of cultivation; however, following a wheat crop withoutstraw removal, direct drilled wheat had a significantly higher DON level compared to wheatfrom plots which were either ploughed or min-tilled [125].

In accordance with the guidelines contained in the Codex Alimentarius, soil should betested to determine if there is need to apply a fertilizer and/or soil conditioners to assureadequate soil pH and plant nutrition to avoid plant stress, especially during seed devel‐opment [122].

Research of [126] showed that supplementary nitrogen and a plant growth regulator in‐creased, by up to 125%, the incidence of infection by Fusarium species in the seed of wheat,


barley and triticale. Similarly, in the studies of [127], a significant increase in fumonisins anddeoxynivalenol contamination in the grain of wheat and kernels was observed with increas‐ing N fertilizer from 0 to 80 kg/ha. That research concluded that in practical crop husbandry,Fs cannot be sufficiently controlled by only manipulating the N input [111]. The study of[128] showed that the use of six different combinations of agricultural practices (sowingtime, plant density, N fertilization and European corn borer (ECB) control with insecticide)can effectively lead to good control of fumonisins and deoxynivalenol in maize kernels.

7.4. Use of chemical and biological agents

In accordance with the guidelines contained in the Codex Alimentarius [122], farmersshould minimize insect damage and fungal infections of the crop by proper use of registeredinsecticides, fungicides and other appropriate practices within an integrated pest manage‐ment program.

Some studies have been conducted to examine the effectiveness of the fungicides which areapplied during flowering can reduce Fusarium infections and subsequent DON in the har‐vested grains. The results of [129] provided that azoles, tebuconazole, metconazole and pro‐thioconazole significantly reduced the Fusarium disease symptoms and Fusarium mycotoxinconcentrations. The greatest reduction in the DON concentration occurred with prothioco‐nazole (10-fold). Azoxystrobin had little impact on the mycotoxin concentration in the har‐vested grain infected by Fusarium species, but could increasing the mycotoxin concentrationin grains when F. nivale was the predominant species present [130, 131]. Fungicide mixturesof azoxystrobin and azole resulted in a lower reduction of DON, compared to azole alone[120, 132]. A number of trials in Germany have indicated that some strobilurin fungicidesapplied before anthesis can also result in increased DON compared to unsprayed plots[133]. Reductions in DON observed in field experiments using fungicides against natural in‐fections of Fusarium are lower and inconsistent [134]. This is probably due to the fact thatduring a natural infection, the infection occurs over a longer period of time.

Alternatively, a limited number of biocompetitive microorganisms have been shown usefulfor the management of Fusarium infections [111]. Research has demonstrated the successfuluse of bacteria in biocontrol of mycotoxigenic fungi. One bacterium, Enterobacter cloacae wasdiscovered as an endophytic symbiont of corn [135]. Corn plants with roots endophyticallycolonized by these bacteria were observed to be fungus-free and in vitro control of F.verticil‐lioides and other fungi with this bacterium was demonstrated. An endophytic bacterium, Ba‐cillus subtilis showed promising for reducing the mycotoxin contamination withF.verticillioides during the endophytic growth phase [136]. Yeast antagonists such as Crypto‐coccus nodaensis were isolated from wheat anthers. The antagonists reduced Fusarium headblight severity by up to 93% in greenhouse and by 56% in field trials when sprayed ontoflowering wheat heads [137]. The most successful antagonists reduced the DON content ofgrain more than 10-fold in greenhouse studies [138].

Actions to be taken during harvest in order to reduce the risk of mould contamination andmycotoxin production include [112]:


209

• harvest as quickly as possible

• avoid field drying

• transport the crop to the homestead as soon as possible

• if lack of labour force or time prevents removal from the field, then dry the crops on plat‐forms raised above ground (if climate is hot and the drying crop can be left to stay on thefield on a platform or cut and tied into stooks) to dry

• bundles of stover should also be placed on platforms to dry and not left lying on the soil

The post-harvest strategies include improving the drying and storage conditions togetherwith the use of chemical, physical or biological methods.

8. Methods of removing mycotoxins from cereals

When mycotoxin prevention is not satisfactory, some decontamination methods are needed.The use of detoxification methods is allowed only in the case of feed and feed components.Foodstuffs containing contaminants exceeding the maximum levels should not be placed onthe market either as such, in the form of a mixture with other foodstuffs or used as an ingre‐dient in other foods. Food contaminated with mycotoxins is not safe for consumers and nodecontamination methods can be used.

According to FAO [111, 139, 140] the feed decontamination process must:

• destroy, inactivate or remove mycotoxins

• not produce toxic, carcinogenic or mutagenic residues in decontaminated final products

• not decrease the nutritive value and organoleptic properties

• destroy all fungal morphological forms

• not significantly increase the cost of production

There are some physical methods of decontamination of feed components such as sort‐ing grains, washing procedures, gamma radiation and UV treatment and also extractionwith organic solvents. These methods are summarized by [140]. Physical removal ofdamaged, mouldy or discoloured kernels significantly decreased the concentration of AFin peanuts. Sorting is not effective for maize and cottonseed. Washing with water or so‐dium carbonate solutions could decrease the concentration of DON, ZEA and fumonisinsin wheat and maize.

High temperature is not used for decontamination of agricultural products, due to thermo‐stability of mycotoxins. Different types of radiation were tested for mycotoxin detoxifica‐tion, but the results were not effective enough.

Chemical compounds such as organic acids, ammonium, sodium hydroxide, hydrogen per‐oxide, ozone, chloride and bisulphite were tested for their efficacy in mycotoxin decontami‐


nation [141, 142]. Chemical decontamination is very effective, but these methods areexpensive and affect the feedstuff quality. Among the chemical methods, only peroxide andammonia are mostly used for aflatoxin removal from feed. Ammoniation works by irreversi‐bly converting AFB1 to less toxic products such as AFD1 [143]. Data show that treatment ofmaize contaminated with 1000 or 2000 ppb aflatoxins with 1% of aqueous ammonia for 48 hremoved 98% of the aflatoxins. There was no significant change in the dietary intake, bodyweight gain, and feed conversion ratio in chickens fed with ammonia-treated aflatoxin-con‐taminated maize, whereas these parameters were suppressed in birds fed with aflatoxin-containing diet [142]. Atmospheric ammoniation of corn does not appear to be an effectivemethod for the detoxification of F.moniliforme–contaminated material. In the research of[144], the levels of fumonisin B1 in naturally contaminated corn were reduced by about 45%due to the ammonia treatment. Despite this, the toxicity of the culture material in rats wasnot altered by ammoniation.

A recent and promising approach to protect animals against the harmful effects of mycotox‐in-contaminated feed is the use of mycotoxin binders (MB). They are added to the diet inorder to reduce the absorption of mycotoxins from the gastrointestinal tract and their distri‐bution to blood and target organs. These feed additives may act either by binding mycotox‐ins to their surface (adsorption), or by degrading or transforming them into less toxicmetabolites (biotransformation). Various inorganic adsorbents, such as hydrated sodiumcalcium aluminosilicate, zeolites, bentonites, clays, and activated carbons, have been used asmycotoxin binders. The use of mycotoxin binders is discussed in some review articles[145-147]. The best aflatoxin adsorbent seems to be HSCAS (hydrated sodium calcium alu‐minosilicate), which rapidly and preferentially binds aflatoxins in the gastrointestinal tract[148-150]. The prevention of aflatoxicosis in broiler folders was examined by [150]. HSCASand activated charcoal were incorporated into the diets for broilers containing purified afla‐toxin B1 (7.5 ppm), or natural aflatoxin produced by Aspergillus parasiticus on rice (5 ppm).The authors showed that HSCAS significantly decreased the growth-inhibitory effects ofAFB1 or AFs on the growing chicks, namely by 50 to 67%. The authors suggest that HSCAScan modulate the toxicity of aflatoxins in chickens; however, adding activated charcoal tothe diet did not appear to have protective properties against mycotoxicosis [150].

Physical and chemical methods have a lot of disadvantages; in many cases they do not meetthe FAO requirements. Therefore, the use of other methods is considered. Biological meth‐ods, involving decontamination with microorganisms or enzymes, give promising results.Recently, an increase in the research connected with mycotoxin detoxification by microor‐ganisms has been observed. Several studies have shown that some bacteria, moulds andyeasts such as Flavobacterium auriantiacum, Corynebacterium rubrum, lactic acid bacteria (Lac‐tobacillus acidophilus, L.rhamnosus, L.bulgaricus), Aspergillus niger, Rhizopus nigricans, Candidasp., Kluyveromyces sp., etc. are able to conduct detoxification of mycotoxins (Tab. 6). Unfortu‐nately, few of these findings have practical application.

Already in 1966, a review of microorganisms was conducted by [151] as for their capabilityof degrading aflatoxins. It was found that yeasts, actinomycetes and algae did not show thistrait, but some moulds, such as Aspergillus niger, A. parasiticus, A. terreus, A. luchuensis, and


211

Penicillium reistrickii, partially transformed aflatoxin B1 to a new product. Among them, onlythe bacteria Flavobacterium aurantiacum (now Nocardia corynebacterioides) is able to removeaflatoxin, both from the media and from the natural environments such as milk, oil, cocoabutter and grain. It was shown that to obtain the apparent loss of the toxin, it was necessaryto use the bacterial population with the density of more than 1010 CFU/ml [154, 188].

Mycotoxin Microorganism References

Aflatoxin B1 Flavobacterium aurantiacum (Nocardia corynebacterioides),

Lactobacillus acidophilus, L.johnsonii, L.salivarius, L.crispatus, L.gasseri,

L.rhamnosus, Lactococcus lactis, Bifidobacterium longum, B.lactis,

Mycobacterium luoranthenivorans, Rhodococcus erythropolis, Bacillus

megaterium, Corynebacterium rubrum, Kluyveromyces marxianus,

Saccharomyces cerevisiae, Aspergillus niger, A. terreus, A.luchuensis,

Penicillium reistrickii, Trichoderma viride

[151-165]

Ochratoxin A Lactococcus salivarius subsp. thermophilus, Lactobacillus delbrueckii

subsp. Bulgaricus, L. acidophilus, Bifidobacterium animalis, B. bifidum,

Lactobacillus plantarum, L. brevis, L. sanfranciscensis, L.acidophilus,

Acinetobacter calcoaceticus, Rhodococcus erythropolis, Oenococcus

oeni, Saccharomyces cerevisiae, Kluyveromyces marxianus, Rhodotorula

rubra, Phaffia rhodozyna, Xanthophyllomyces dendrorhous,

Metschnikowia pulcherrima, Pichia guilliermondii, Trichosporon

mycotoxinivorans, Rhizopus sp., Aureobasidium pullulans, Aspergillus

niger, A.carbonarius, A. fumigatus, A. versicolor

[166-183]

Fumonisin B1 Lactobacillus rhamnosus, Lactococcus lactis, Leuconostoc mesenteroides,

Saccharomyces cerevisiae, Kluyveromyces marxianus, Rhodotorula rubra

[176, 184]

Trichotecenes Ruminant bacteria, chicken intestinal microflora,

Saccharomyces cerevisiae, Kluyveromyces marxianus, Rhodotorula rubra

[176, 185, 186]

Zearalenone Soil bacteria, Propionibacterium fraudenreichii, Rhizopus sp.,

Trichosporon mycotoxinivorans

[179, 183, 187]

Table 6. Decontamination abilities of microorganisms

It was observed that cultures of toxinogenic Aspergillus flavus and Aspergillus parasiticus wereable to reduce aflatoxin contamination. Aflatoxins were degraded by the strains that pro‐duce them, but only after the fragmentation of the mycelium. The cause of this phenomenonwas absorption into the cell wall of mycelium [165]. In the research of [176], 10 yeast strainsof the Saccharomyces, Kluyveromyces and Rhodotorula genera were studied for their ability toperform biodegradation of fumonisin B1, ochratoxin A and trichothecenes. Significant differ‐ences were demonstrated between the strains, but there were no preferences as to the typesof mycotoxins. Fumonisins were removed by the majority of the strains in 100%, the remov‐al rate for deoxynivalenol ranged from 63 to 100%, and for ochratoxin A from 69 to 100%.The possibility of using moulds to remove ochratoxin A was studied by [179, 182]. The au‐


thors selected two out of 70 isolates of the Aspergillus species - Aspergillus fumigatus and As‐pergillus niger, which transformed ochratoxin A to ochratoxin α and phenylalanine within 7days of incubation on both liquid and solid media.

In vitro studies conducted by [186] demonstrated the degradation of 12 trichothecene myco‐toxins conducted by bacteria isolated from the digestive tract of chickens. The transforma‐tion of the toxin led to their partial or total deacylation and de-epoxidation. Similarly, it wasshown, that the strains of anaerobic bacteria - isolated from the rumen, Gram positive, pre-classified to the genus Eubacterium - are able to perform the transformation of type A tricho‐thecenes to non-toxic forms [185].

The above-presented examples of microbial activity aimed at removal of mycotoxins aremainly of scientific nature, allowing for a better understanding of the strains, their proper‐ties and the mechanisms of the processes. Their limited practical application made that re‐search turned in the direction of such organisms, which can be used in biotechnologicalprocesses during production, such as fermented food production, where the raw materialmay be contaminated with mycotoxins. The most important among them are lactic acid bac‐teria and yeasts Saccharomyces cerevisiae [163].

Literature data indicate the existence of strains of lactic acid bacteria with different abilitiesto remove mycotoxins, as demonstrated both in in vitro and in vivo studies conducted byvarious authors with the use of some strains of probiotic Lactobacillus rhamnosus, Lactobacillusacidophilus, Bifidobacterium bifidum, B. longum, and Streptococcus spp., Lactococcus salivarius,Lactobacillus delbrueckii subsp. bulgaricus [155, 156, 158, 160, 169, 189, 190]. According to [191],the decontamination process is very fast; after 4h the toxin concentration was reduced from50 to 77%. It was observed that heat-inactivated cells were more effective than living cells,which results from the changes in the surface properties of cells, which occur under hightemperature [191]. The capacity to reduce the content of ochratoxin A in milk by lactic acidbacteria belonging to the species Lactococcus salivarius, Lactobacillus delbrueckii subsp. bulgari‐cus and Bifidobacterium bifidum was confirmed in [167]. The content of patulin in the mediumdecreased in the level from 10 to 82% under the influence of bacteria belonging to the genusLactobacillus and Bifidobacterium. The decontamination process depends on the inoculumdensity, pH and the concentration of toxins. Among the studied strains, L.acidophilus, re‐moves up to 96% of the toxin added to the medium in an amount of 1ppm [166].

Our in vivo experiments indicate that the use of probiotics as feed additives limited the ef‐fects of mycotoxins in animals, as well as reduced the accumulation of toxins in the tissues,thus reducing the contamination of food of animal origin with the toxins [192]. It was shownthat Lactobacillus rhamnosus bacteria limited by 75% the adsorption of aflatoxin B1 in the di‐gestive tract of chickens [189].

The second group of organisms with a potential application in detoxification is constituted bySaccharomyces cerevisiae yeasts. Our own research demonstrated that these organisms are capa‐ble of eliminating ochratoxin A from the plant raw material during fermentation and chroma‐tographic analysis did not show any products of OTA metabolism, which proves that it wasnot the case of biodegradation. The amount of ochratoxin A removed by bakery yeasts after 24-


213

hour contact equalled from 29% to 75% for 5 mg d.m/ml and 50 mg d.m./ml, respectively. Theprocess of adsorption proved to be very fast; immediately after mixing the cells with the toxinits amount significantly decreased, and lengthening the contact up to 24 hours did not bringfurther notable changes. The presence of physiologically active cells is not necessary in order toremove the toxin; the dead biomass also removed OTA from the buffer and the amount of thetoxin removed was much bigger than in the case of the active biomass. In the case of the 5mg/ml density, 54% of the toxin was adsorbed, i.e. twice more than in the case of the active bio‐mass [171]. The reason for OTA removal was adsorption of the toxin to the yeast cell wall. Thismechanism was independent of the type of toxin, as demonstrated in relation to aflatoxin B1,zearalenone and T-2 toxin and patulin. The compounds of the cell wall that are involved in thebinding process are probably β-D-glucan and its esterified form [193, 194]. Yeasts and their cellwall components are also used as feed additives for animals, and as adsorbents, which effec‐tively limits mycotoxicosis in farm animals [195, 196].

The potential application of yeasts as adsorbents for foods and feeds depends on the stabili‐ty of the toxin binding to the cells in the conditions of the gastrointestinal tract. According to[194], zearalenone adsorption is most effective at a pH close to neutral and acidic, and there‐fore those which prevail in some regions of the gastrointestinal tract. The result of the use ofyeasts to remove ochratoxin A is detoxification of the environment, as demonstrated in thecytotoxicity and genotoxicity tests using pig kidney cell lines [197]. Some yeasts also exhibitfeatures of probiotic activity, which is an additional argument for the use of these organisms

The use of microorganisms or their cell components for decontamination of foods andfeeds has raised high hopes, but also the controversy from the perspective of the con‐sumer. There are no legal regulations devoted to this issue, and the data referring to thestability of the microorganism-toxin connection in the gastrointestinal tract, as well astoxicological data are still incomplete. The only group of microorganisms, which in addi‐tion to other advantageous features of health promotion has the ability to remove toxins,is probably that of probiotic lactic acid bacteria. Also, Saccharomyces cerevisiae yeast andits cell wall component - glucan can be used for this purpose. These factors can be ap‐plied both as human dietary supplements and ingredients in animal nutrition, as well asduring biotechnological processes.

Author details

Małgorzata Piotrowska1*, Katarzyna Śliżewska1 and Joanna Biernasiak2

*Address all correspondence to: [email protected]

1 Technical University of Lodz, Faculty of Biotechnology and Food Sciences, Institute of Fer‐mentation Technology and Microbiology, Lodz, Poland

2 Technical University of Lodz, Faculty of Biotechnology and Food Sciences, Institute ofChemical Technology of Food, Lodz, Poland


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